Chronic ethanol consumption causes immunosuppression, particularly related to Th1 cytokine expression, and causes increased susceptibility to infection including infection with the intracellular bacterial human pathogen, Listeria monocytogenes. CpG DNA induces innate Thl-like responses and reduced susceptibility to Listeria monocytogenes detected by decreased replication of L. monocytogenes in murine spleens and livers. CpG immunostimulatory DNA will be used to stimulate innate systemic and mucosal immune responses in ethanol- fed and water-fed control mice. Induction of cytokine expression in vitro and in vivo in spleen and Peyer's patch cells from CpG-treated, ethanol-fed and water-fed control mice will be detected by enzyme-linked immunosorbant assay (ELISA), and specific Thl cytokine expressing cells will be identified by magnetic cell sorting (MACS) and intracellular cytokine staining. CpG DNA treatment and immunostimulation will be used to overcome ethanol-induced immunosuppression, which will be detected by reduced replication of L. monocytogenes in spleens and livers by a colony forming (CFU) assay. Various routes and doses of CpG treatment and challenge with L. monocytogenes including intraperitoneal (IP), intravenous (IV), and oral will be used to elicit both systemic and mucosal immunity and to determine the limits of protection induced by CpG DNA. Reduced replication of L. monocytogenes will be correlated with cytokine expression detected by ELISA and the mechanism of CpG-induced reduction in replication will be determined by identifying by flow cytometry and intracellular cytokine staining whether specific cells may be suppressed or depleted by chronic ethanol consumption and stimulated by CpG treatment. Because defects in killing Mycobacterium avium, an intracellular bacteria similar to L. monocytogenes and to the organism that causes tuberculosis, have been demonstrated for macrophages following exposure to alcohol, intracellular killing of L. monocytogenes by peritoneal macrophages will be examined to determine whether ethanol-fed mice are deficient in killing L. monocytogenes and whether CpG DNA stimulation may enhance killing. These studies could lead to treatments for protecting alcoholic patients from intracellular bacterial infections such as those caused by L. monocytogenes or Mycobacterium tuberculosis (TB).